The present invention relates to Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS). This method introduced by Karas and Hillenkamp in 1988 has become established as a method for mass determination of biopolymers and substances such as peptides, proteins and DNA fragments. In this method, the substance to be analyzed is typically placed in a solution of matrix material and coated onto a support. The solute evaporates, leaving the analyte in a solid matrix which is then illuminated to cause the analyte molecules or synthetic polymers to be desorbed. This desorption process is especially useful for releasing large biological molecules without charring, fragmentation or chemical degradation to a mass spectrometer or similar instrument for separation and detection.
In common MALDI mass spectrometry setups, the sample is placed on a metal substrate and is irradiated from the side facing the ion analyzer ("top illumination"). In contrast to this arrangement, transmission illumination is also possible wherein the rear side of the sample is illuminated through a transparent substrate, for example by a focused laser beam. In either event, the matrix consisting of a relatively low molecular weight aromatic organic substance such as nicotinic acid, benzoic or cinnamic acid, holds a quantity of the peptide, protein or other substance to be analyzed and is irradiated with a high intensity laser beam to cause desorption of the analyte from a surface of the sample. In a representative mass spectrometry arrangement, ions of the analyte are subjected to electric field acceleration for mass separation in the instrument, such as a time-of-flight (TOF) mass spectrometer. The laser radiation is selected such that matrix strongly absorbs at the laser wavelength and desorbs material sufficiently quickly to form an ejecting plume at its outer surface. The laser wavelength, however, is sufficiently long to not break bonds of the analyte, and the desorption process is non-thermal so that large molecules are provided to the spectrometer instrument substantially intact. The mechanism of desorbing or ejecting large molecules from a relatively light matrix material is quite general, and allows one to detect analytes having a mass of tens or hundreds of kiloDaltons. Various techniques for preparing certain classes of substances for desorption from a variety of matrix materials have extended the usefulness of the technique.
In conventional instruments, the standard configuration involves performing both illumination and mass analysis from the same side of the sample. This produces a relatively high yield of large molecule ions and good mass resolution. Illumination spot sizes of between 50 and 1000 micrometers in diameter have been used with illumination levels in the range of 10.sup.6 -10.sup.7 watts/square centimeter to essentially eject small volumes of the sample and provide a short, high velocity plume of material for analysis. An extensive library of organic large molecule spectra has been built up using such instruments. Nonetheless, the geometrical requirements of providing an ion extraction and acceleration optics in a vacuum flight chamber with a number of high voltage electrodes to accelerate the material to an analysis detector impose constraints on the optical path of the laser illumination beam, resulting in a relatively costly and inflexible instrument. Furthermore, the provision of a relatively high energy plume as the initial ion source results in a spread of velocities and spatial position of the initial burst of ions, which in addition are subject to differing electric fields because of the spatial spread of the plume, so that, while the instrument provides a good yield of analyte, the mass resolution is compromised. In general, the matrix molecules provide an internal reference peak. However, since the analyte is often many times greater in mass, and the mechanism of desorption is also not fully understood, it is also possible that the acquired spectra include unrecognized shifts and other artifacts resulting from initial plume geometry or release dynamics that will complicate the accuracy of mass determination and a comparison with independently produced spectra in the future.
Various researchers have explored transmission MALDI for different materials and one or more matrix compositions, and have been able under some conditions to obtain results analogous to those accumulated using the more prevalent top side illumination. In general, by separating illumination and the mass analysis instrumentation on opposite sides of a matrix one might expect to implement different instrument designs with greater freedom. In particular, scanning arrangements might be implemented to allow the selective analysis of particular spots or sample locations. Furthermore, the ability to provide ion desorption from a defined surface may be expected to yield sharper spectra. However in switching from a top illumination configuration to a transmission illumination configuration, one of necessity changes the nature of a number of essential processes underlying the desorption technique. Thus for example the shape of the plume, the velocities or directions of molecules or ions upon release, and the underlying mechanism or yield of the release may all be affected by a change in the illumination/desorption geometry. Relatively few experiments have utilized transmission illumination, and these have in general yielded lower quality spectra than top illumination, and have been tried only with a limited range of relatively light analyte molecules.
The essential mechanisms by which material is desorbed are not fully understood, and effects may vary with different materials. In general, to make a measurement one tunes the analyzer by setting appropriate electric and/or magnetic fields, or otherwise defining a sample window, then illuminates the matrix, progressively increasing the fluence until the spectrometer starts to detect desorbed ions. The fluence may generally then be increased somewhat to increase the amount of the heavy analyte present in the desorbed material, but should not be increased so much as to introduce charring or fragmentation of the material. In general, increase in illumination fluence increases the amount of material released. However, as described above, the mass resolution, which is initially limited, may suffer due to an increased spread of initial velocities, the irregular geometry of the emission plume or other factors.
It is therefore desirable to provide a transmission MALDI method or apparatus in which resulting spectra are identical to or well correlated with MALDI spectra obtained in top illumination of similar compounds.
It is also desirable to provide new transmission MALDI stages or mechanisms for desorption of an analysis sample.
It is also desirable to provide a MALDI spectrometry process wherein the peaks are improved, and mass resolution refined.